Objective:

Prenatal exposure to the endocrine disruptor tributyltin (TBT) is known to promote adiposity in mice; mesenchymal stem cells (MSCs) harvested from these animals are biased towards the adipose lineage. It is known that TBT promotes adipose differentiation through activation of two nuclear receptors: Peroxisome proliferator-activated receptor gamma (PPARgamma) and Retinoid X receptor (RXR). This research investigates the role of TBT in the often overlooked process of lineage commitment--which precedes differentiation--and seeks to understand how TBT commits undifferentiated MSCs to an adipose fate.

Approach:

To understand how TBT can promote adipose lineage commitment of MSCs I have developed an in vitro assay to screen for EDCs that can affect the commitment process. Using this new assay I will assess if TBT acts through PPARgamma and/or RXR to promote adipose lineage commitment using pharmacologic activators and/or inhibitors of these nuclear receptors. Furthermore, I will use a deep-sequencing approach to assess how TBT alters the transcriptome and epigenome of undifferentiated MSCs. In addition to samples treated with TBT and vehicle control, sequenced samples will include cells treated with a selective PPARgamma agonist (Rosiglitazone) and RXR agonist (AGN194204). These treatments will help elucidate which nuclear receptors and gene targets TBT acts through to commit MSCs to the fat lineage.

Expected Results:

Preliminary data from our in vitro assay suggest that TBT commits MSCs to the adipose lineage through an RXR-dependent, PPARgamma-independent mechanism. We expect our deep-sequencing studies to reveal many novel targets of TBT. Given our RXR-dependent phenotype, we expect genomic targets shared by TBT and RXR agonist to be good candidates for further investigation. In addition, genome-wide analysis of the epigenome will be integrated with RNA-seq data to reveal genomic targets where TBT alters epigenetic marks to influence gene expression. Top gene candidates will be investigated using lentiviral knockdown/overexpression, pharmacologic inhibition/activation when possible, and an assessment of direct RXR binding in neighboring regulatory regions.

Supplemental Keywords:

Progress and Final Reports:

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